In fluorescence microscopy, light of different wavelengths is needed (i) for exciting various fluorescent dyes for fluorescence, (ii) for purposefully de-exciting various fluorescent dyes again (for example in STED fluorescent light microscopy), (iii) for purposefully transferring various fluorescent dyes into a dark state (for example in GSD fluorescent light microscopy), or (iv), within overlapping but different spatial areas, for exciting a dye for fluorescence, on the one hand, and for de-exciting the same dye by stimulated emission or for transferring the same dye into a dark state, on the other hand. To meet these demands, a plurality of different monochromatic light sources may be used, each of which provides monochromatic light of one of the various wavelengths. As the monochromatic light sources suitable to this end are, as a rule, lasers of high quality, because the light is needed in pulses of high intensity, fluorescent light microscopes designed in this way are very expensive. For some wavelengths, it is even difficult to find suitable lasers of sufficient quality.
Inter alia from German Patent Application published as DE 10 2005 020 003 A1 and from corresponding International Patent Application published as WO 2006/114247 A1 is known to use a gas laser as a light source in fluorescent light microscopy, which displays a plurality of useable emission lines or which is at least tunable to a plurality of emission lines. Gas lasers, however, are expensive to purchase and costly in operation. In addition, in a gas laser, the suitable emission lines at best cover a small spectral range in such a way that the absorption lines of potential fluorescent dyes falling within this spectral range may be addressed at a high effective cross-section.
In a fluorescent light microscope which is known from U.S. Pat. No. 6,710,918, light of a certain wavelength used for transferring a fluorescent dye in a sample from one state into another state is provided by injecting light of another wavelength from a laser, particularly a mode-coupled titanium sapphire laser which emits at a wavelength of 800 nm, into an optical element which distributes the intensity of the injected light of one wavelength over a continuous spectrum which comprises shorter and longer wavelengths than the injected light and which is referred to as a supercontinuum. Here, the optical element may, for example, be an optical wave guide fiber strongly tapering in cross-section over a length of about 30 mm to 90 mm (a so-called “tapered fiber”), which has an overall length of about 1 m, or a micro-structured optical fiber with a photonic band gap, which is also referred to as a photonic crystal fiber. In a photonic crystal fiber, the photonic band gap is produced by a honeycomb-like microstructure around a very small fiber core of just about 2 μm diameter. The typical length of a photonic crystal fiber (PCF) is 38 cm. The continuous spectral distribution allows for selecting light of any wavelength for transferring a fluorescent dye from one state into another from the supercontinuum. The light power available at each single wavelength of the supercontinuum, however, is extremely reduced as compared to the output power of the pumping laser. Further, considerable efforts are to be taken to provide a supercontinuum having an at least approximately homogenous intensity distribution and, particularly, an intensity distribution which is stable in time. Thus, the development of corresponding fluorescent light microscopes from the patent application of this idea up to commercial products has taken more than five years. Further, the corresponding fluorescent light microscopes are expensive which is inter alia due to the titanium sapphire laser having the required output power and the PCF used in the commercial product.
In a light source for fluorescent light microscopy, including STED microscopy, which is known from German Patent Application published as DE 103 47 712 A1 and from corresponding U.S. Pat. No. 7,433,119, the wavelength of light is controlled by means of a photonic fiber or an opto-parametric oscillator (OPO). In an OPO the desired wavelength is generated by means of an optical non-linear three-wave frequency conversion process in which the input frequency of a pump wave is divided into two frequencies (signal and idler).
In a Raman amplifier arrangement on the basis of a standard single-mode fiber which is known from German Patent Application published as DE 100 12 881 A1 and from corresponding U.S. Pat. No. 6,870,980, Stokes waves are generated by means of stimulated Raman scattering of a high power optical pump signal to amplify an incoming optical signal.
A device by which incoming light is spectrally split-up based on dispersion and polarization effects is described in European Patent Application published as EP 1 662 296 A1 in corresponding US Patent Application published as US 2006176542 A1. This device is also proposed as a light source for use in STED microscopy, wherein light of a wavelength, which exactly corresponds to the wavelength of the fluorescent light of interest from a sample so that this light is particularly well suited for de-exciting by means of stimulated emission, is separated from the incoming laser beam, whereas the remaining light of the laser beam is used excitation light.
A device for generating light in a desired wavelength range, by which the excitation of a sample shall be possible in an even smaller spatial area than in STED microscopy, is known from International Patent Application published as WO 03/016781 A2 and corresponding U.S. Pat. No. 7,318,907. Here, the light emission of the device is based on photo-induced excitation of surface plasmons in a metal layer.
There still is a need for a fluorescent light microscope for measuring a sample, in which light of different wavelengths is available to transfer various fluorescent dyes from one state into another state without high and costly efforts and without stability problems.